EVOGENE Seminar - Christoffer Bugge and Marcin Michalik
This seminar will consist of two 20 minutes talks entitled:
"Identification and evaluation of peptides for active and passive immunization against Pseudomonas aeruginosa"
"Mycena - hidden biotrophic interactions"
"Mycena - hidden biotrophic interactions" presented by Christoffer Bugge, EVOGENE
Abstract: My Marie Curie-funded project concerns the life strategy of Mycena (or bonnet mushrooms). As all my work is still in progress, I will present the project as an outline and briefly discuss some preliminary results and promising indications.
Mycena is a widespread group of macrofungi with important functions in forests and other terrestrial ecosystems. While its members have traditionally been uniformly assumed to be degraders of litter, wood and other dead biological matter, very recent research has suggested that several members could in fact be parasitically or mutualistically biotrophic with plants. In numerous studies, environmental sequences with affinity to Mycena have been observed associated with plant roots. We employ a multifaceted, interdisciplinary approach where we combine traditional knowledge about fungal cultivation with advanced DNA sequencing technology and isotope analyses techniques. The rapidly developing high throughput DNA sequencing technology (HTS) produces millions of DNA strands within hours, and by applying this to plant roots, we will quickly obtain an overview of the presence of Mycena in or on plant roots (indicating a possible biotrophic relationship). We will create a database of Mycena sequences from our own sequencing as well as sequences already publicly available. By sequencing the entire genome of five carefully selected species of Mycena, we will be able to analyse the genomic content associated with different life strategies. As molecular identification of Mycena associated with plant roots does not indicate an ecological interaction per se, we will employ innovative research techniques designed to quantify potential interactions. Isotopic fractionation of carbon/nitrogen of Mycenas and plants at field sites can reveal the mushroom´s source of carbon, and co-culturing of biotrophic Mycenas together with plants will provide direct evidence for the nature of their relationship(s). This research will clarify the ecology of a prominent and widespread genus of fungi, thereby illuminating its role in forest and general plant ecology.
"Identification and evaluation of peptides for active and passive immunization against Pseudomonas aeruginosa" presented by Marcin Michalik, EVOGENE
Abstract: Pseudomonas aeruginosa is a Gram-negative bacterium, commonly present in the environment, that causes serious health problems when it infects humans and is resistant to many antibiotics and treatments. Patient groups at risk for acquisition of P. aeruginosa infections include those with cystic fibrosis (which almost always leads to chronic airway infection), paraplegic, burn, and immunocompomised patients. Prevention of infection by vaccination is desirable, but until today no vaccine has yet obtained market authorization. This project is aimed at the development of a peptide-based P. aeruginosa vaccine.
We have selected short immunogenic peptide epitopes from P. auruginosa surface localized proteins using bioinformatic tools. Our current goal is to insert multiple peptides into a stable protein scaffold. In first trials we obtained stable recombinant proteins which are recognized by antibodies against the inserted peptides. Simultaneously, we are checking if the selected peptides/antigens are present in clinical strains from infected patients’ lungs. We will then evaluate the targets step by step, by immunoblotting of P. aeruginosa preparations, by checking the targets against serum samples of infected CF patients, and by immunofluorescence experiments to verify that the target peptides are indeed surface-exposed and if raised antibodies against peptides can detect the original proteins from which the peptides were selected.
Once the recombinant proteins have been pre-tested for their stability and their epitope display, we would like to obtain polyclonal antisera for the most promising combinations for further testing – as a first step towards phase I clinical trials. The $5000 research grant will be spend to produce rabbit antisera against selected multiepitope proteins which will be produced in E. coli cells and purified by physical methods and anion exchange chromatography.